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Published by faats
pages 1 to 6 ra ni. :)
pages 1 to 6 ra ni. :)

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Published by: faats on Sep 09, 2010
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CYTOCHEMISTRYI.Enzymatic TechniquesA.PeroxidasesA H
O +H
OA.1. Diaminobenzidine MethodReagents: Fixative-buffered formalin-acetoneIncubation mixture: (prepare fresh for each batchPhosphate buffer 0.07M,pH 7.4-50mL3,3 DAB tetrahydrochloride-37.5mg3% H
Giemsa stain, 10.0 mLProcedure:Controls: blood film from normal donorPositive control (neutrophils)Negative controls (lymphocytes)Interpretation: Dark brown granules in the cytoplasm of granulocytesand monocytesMonocyte: weak to moderateGranulocytes: are packed with positive granulesRBCs-stain diffusely brown because of pseudoperoxidase activity in HbA.2. Cyanide-Resistant Peroxidase Stain:Uses: as proposed by Gabbas and Li1.identification of the eosinophilic component of acute myeloid andacute myelomonocytic leukemias2.aid in recognition of de novo acute eosinphilic leukemiaIncubation Medium: In addition to DAB method4.9 mg NaCN (sodium cyanide) is added and titrate to pH 7.4 with 1NHClProcedure: same with DABIncubation mixture-5 minutesControls: Normal blood buffy coatEosinophils should be strongly positive (brown)Neutrophils-negativeB.Esterases: are enzymes that hydrolyze aliphatic and aromaticesters at acid or neutral pH
B.1. Naphthol AS-D Chloroacetate EsteraseUses:1.Useful in demonstrating myeloid elements on paraffin-embeddedsections such as granulocytic sarcoma2.systemic mast cell diseaseHexazotized new fuchsin: mix equal volume of new fuchsin and 4% Nanitrite for 1 min before useNaphthol AS-D chloroacetate solution: 10mg Naphthol AS-Dchloroacetate dissolve in 5mL of N,N-dimethyl formamide.Store at 4°-10°C. Stable for 1 month.Phosphate buffer 0.07M, pH 7.73Mayer’s hematoxylin-10 min. Counterstain.Procedure:3.Incubate films at room temp for 10 minutes using:0.07M phosphate buffer-38.0mLFresh hexazotized new fuchsin-0.2mLNaphthol AS-D chloroacetate sol-2.0mLControls: Normal neutrophils-positive controlInterpretation:B.2. Alpha-Naphthyl Acetate Esterase (ANAE)T-helper lymphocytes-focal dotlike staining with long incubationT-lymphoblasts of acute leukemia-focal staining is weak or negativeHexazotized pararosanilin: Mix equal volume of the first 2 solution for1 minute before use.Phosphate buffer, 0.07M pH 6.64Alpha-naphthyl acetate solu: 100mg dissolve in 5.0mL ethylene glycolmonomethyl ether. Refrigerate before use.Controls:Positive controls: Normal monocytes or histiocytes in bonemarrow or blood film prep.Monocytes-stain red-brownLymphocytes-dotlikeB.3. Alpha-Naphthyl Butyrate Esterase (BE)
Use to differentiate acute myelomonocytic, acute monocytic andchronic myeolomonocytic leukemias from other acute nonlymphocyticleukemias and dysmyelopoietic syndromes.Incubation mixuter: 45mins. at room temp.0.07M phosphate buffer pH 6.69-38.0mLFresh hexazotized pararosanilin-0.4mLAlpha-naphthyl butyrate solution-2.0mLControl: same with ANAEInterpretation: Enzyme activity is noted as dark red precipitates in thecytoplasm of monocytes and histiocytesB.4. Combination of Alpha-naphthyl butyrate-chloroacetate EsteraseProcedure:BE incubation mixture at room temp. for 30 min.CE incubation mixture at room temp. for 5 min.Controls: normal monocytes and neutrophils in blood filmhistiocytes or developing neutrophils is bone marrowInterpretation:Cytoplasm of monocytes&histiocytes-dark red pptNeutrophils-blue granulesC. PhosphatasesC.1. Acid phosphataseReagents:Fixative: methanol-acetone mixture-30 secondsAcetate bufferSubstrate SolutionNaphthol AS-BI phosphoric acid-100.0 mgN,N-dimethyl formamide-10mgStable for 2 months at 4°-10°C45 min.-incubation mixture: 50mL acetate buffer0.5mL substrate5 mg fast garnet GBC saltMayer’s hematoxylin-counterstain-5-20 min.Mounting medium Glycerin jellyControl: peripheral blood filmInterpretation: discrete purplish to dark red granules

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